Dynamics of global chromatin landscape through the cell cycle and differentiation

The genomes of all higher organisms are packaged in a form known as chromatin, a combination of the DNA and proteins that are bound to it. This packaging of DNA into chromatin is essential because each individual chromosome consist of a single molecule of DNA up to several centimeters long but which must to be packaged up in a cell nucleus only a few micrometers across. Indeed the total DNA length in a normal human cell is about 2m. At the same time, the DNA must be accessible when required to allow a controlled expression of genes, or the copying of DNA during the cell division cycle. The basic unit of chromatin packaging is known as the nucleosome, which consists of a core of special highly conserved proteins known as histones around which the DNA is wrapped approximately twice. Nucleosomes appear to be located in specific positions, particularly around important features such as genes. It has become clear that many characteristics of organisms, and some diseases, are affected by so-called epigenetic modifications, resulting from changes in the chromatin or modification of the DNA rather than changes in the underlying DNA sequence itself. Hence understanding chromatin is of fundamental importance in many biological processes.

In addition to nucleosomes, there are many other complexes of proteins that bind to DNA for a number of purposes, particularly the controlled expression of genes. The pattern of nucleosomes and other proteins binding to DNA can be visualised by using enzymes that cut DNA only where no proteins are bound. Regions that are protected are not cut, and digesting chromatin with such enzymes generates many millions of DNA fragments. These are predominantly the size of single or multiple nucleosomes.

We have developed a new technology that can simultaneously determine all such protected regions across the entire genome, and hence map every nucleosome and other DNA-bound protein complex to generate a "chromatin landscape". This makes use of a high throughput sequencing to generate DNA sequence from both ends of around 180 million such fragments simultaneously. This data is then used to map the endpoints of the fragments onto the genome sequence, thus locating the positions of the bound proteins through regions with few cut ends. We have demonstrated this technology using the model plant Arabidopsis. In this proposal will use this new approach to understand how chromatin structure changes both through the cell cycle as cells divide and as cells differentiate to adopt new characteristics. This analysis will be carried out in both cell culture and in cells from intact tissues, allowing a detailed understanding of how chromatin changes occur during these processes. Specifically we will study how chromatin structure in Arabidopsis changes in response to light and how it is different in root and shoot cells. We will also investigate how this pattern is changed in a specific mutant in which chromatin structure is altered and in which differentiation is affected. As a result, we will develop a detailed understanding of the organization of chromatin in a higher organism, and how this is dynamically altered as the fate of cells changes during the processes of development. These techniques and approaches will be applicable to all eukaryotic organisms and will be of great significance in progressing our understanding of the genome.

Packaging of eukaryotic genomes into chromatin and the positioning of nucleosomes affects every process that occurs on DNA, as the nucleosome modifies or occludes access to underlying DNA sequences. We understand little of the changes in chromatin structure and occur in higher eukaryotic cells, for example during the cell cycle or during the processes of differentiation. Almost nothing is known of the chromatin structure of cells in a tissue context in terms either of nucleosome position or of the global changes in DNA-bound transcription factor complexes. We have established, for the 1st time in a higher eukaryote, a technique we call "chromatin landscaping" that allows us to map with base pair resolution the position of all DNA associated protein complexes across the genome. We have developed this for the model higher plant Arabidopsis because of its small genome size and excellent range of genomics tools and data. We will apply this technique first to understanding chromatin changes during the cell cycle in cultured cells. We will correlate this with detailed data on histone modification being obtained by our collaborators in the same cell type. We will then apply it to cells isolated from intact tissues by flow sorting, allowing us to compare mitotic cells and differentiated cells of both roots and shoots. Finally we will compare the chromatin maps between wild-type and a mutant in a transcription factor known to be associated with cell differentiation and to bind to multiple sites across the genome. Chromatin changes will be correlated with chromatin immunoprecipitation data and RNA profiles. This will (1) provide benchmark high resolution maps of nucleosome positioning in key developmental systems in Arabidopsis; (2) provide new understanding of chromatin changes associated with the cell cycle and cell differentiation, (3) establish how changes in differentiation caused by the binding of a specific factor are reflected in chromatin structure.

This research will enhance our understanding of how the genome functions and how key biological processes are controlled. It will also develop and demonstrate new techniques of genome analysis that can be applied to other organisms, including humans and crops, as well as to basic research in other organsisms.

There are three main groups of beneficiaries: Academic research scientists, industry research and development scientists, the general public and schools.

Academic research scientists will benefit from a greatly increased understanding of genome structure and function. More specifically, a detailed map of chromatin will be developed which will provide the benchmark for all future work in this field. The map will locate the positions of nucleosomes, the basic particle of DNA packaging, as well as other protein complexes bound to the DNA. Importantly as a result of a close collaboration with two groups in the US, this map will be integrated with maps of modifications to the histone subunits of the nucleosomes. In addition to the benchmark map, a detailed understanding of how this varies in different conditions and cell types will be generated. For example, plant cells respond in a profound and general way to light, and the changes that occur in cells in response to light will be defined. When DNA is replicated as cells divide, profound changes may also take place- these are currently unknown and will be defined. Finally as cells take on specialized characteristics and differentiate, it has been known for many years that changes occur in their chromatin, but these have not yet been defined across the whole genome.

Both academic research scientists, and industry based research scientists will benefit from the new techniques developed to analyse chromatin and from easy-to-use software pipelines that can then be applied in other organisms and systems. Hence the technology can be applied in crops or humans to generate a much more detailed understanding of the epigenome- differences that affect how genes are expressed, and which can have profound effects on crop growth or disease responses in humans. Modification of epigenetics has the potential to serve as a new route to the treatment of human diseases and epigenetic modifications are also key mediators of cell fate, with implications for cellular therapies. Understanding chromatin organization is thus of key importance in important applied areas, and chromatin particle spectrum analysis as developed in this grant can make an important contribution. These applications can help improve commercially relevant research in epigenome related areas, in which the UK is a global leader through the research activities of companies such as Pfizer.

More generally this research integrates computational approaches and biology. Significant training potential is foreseen for the two PDRAs. PDRA1 will gain expertise in a broad spectrum state-of-the-art genomics techniques including flow sorting, chromatin analysis, ChIP and RNA Seq with an understanding of the bioinformatics pipeline. PDRA2 will be a computer scientist gaining an understanding and skills for biological application. The development of a skilled workforce with the ability to work collaboratively across discipline boundaries is central to UK Government and European strategies for building the future knowledge based bio-economy, and thus contributes both to UK competitiveness and wealth creation potential.

The general public, and school pupils in particular can benefit from this research. It is easy to understand that since the total DNA content of a human cell is around 2m in length and is enclosed in a nucleus of a few micrometers. Understanding how genes are organized provides an accessible way to discuss the problem of how we can investigate this problem. Engaging with school pupils will provide the staff employed on the project with new didactic and presentation skills.